The present invention features a method of transmitting biologically active materials into cells. More particularly, the present invention features a method of encapsulating a biologically active material in a non-phospholipid vesicle, delivering the vesicle to a cell, allowing fusion between the vesicle and the cell and allowing the biologically active material to diffuse into the cell, thereby transmitting the biologically active material into the cell. The lipid vesicle protects the encapsulated material from extracellular inactivation and, by fusing to the outer membrane of the target cell, delivers the encapsulated material directly into the cytoplasm of the cell. Upon fusion with the cell, material associated with the bilayer of the non-phospholipid vesicle is incorporated directly into the target cell membrane. Accordingly, the present invention also features a method of transmitting bilayer-associated material to a cell.
The transmission of biologically active materials to cells is an essential component of a wide range of therapies. Such therapies include supplying a cell with a protein having a necessary enzymatic activity, providing a new DNA molecule to a cell (gene therapy), immunizing a subject against a foreign protein (vaccination), immunizing a subject against a foreign protein by introducing the gene encoding the protein (gene vaccination) and inhibiting the production of a protein in a cell by providing the cell with a nucleic acid molecule which is antisense to mRNA encoding the protein or otherwise interfering with the mRNA encoding the protein. While the introduction of a biologically active material into a cell is often desirable, there are several obstacles to overcome in order to accomplish this. Transmission of a biologically active material to a cell involves transferring the material from an extracellular site to an intracellular site while maintaining the activity of the material and not damaging the target cell. The phospholipid bilayer that comprises much of the outer membrane of a cell prohibits the indiscriminate entry of materials into the cell. Although certain hydrophobic molecules can passively diffuse through the outer membrane into the cell cytoplasm, most materials encountered by a cell cannot freely enter the cell. Transporter proteins, which form "channels" through the cell membrane, allow passage of certain specific molecules, usually small molecules, into the cell (e.g., ion channels). Cells also express surface receptors, generally in the form of integral membrane proteins, which bind specific ligands and allow their entry into the cell. Molecules which bind to specific cell-surface receptors generally enter the cell via receptor-mediated endocytosis. Other extracellular material can be taken up by a cell by non-specific endocytosis (e.g., pinocytosis). However, materials which enter the cell via an endocytic pathway generally merge with lysosomal vesicles which contain degradatory enzymes. Thus, materials entering the cell by this route are often destroyed or altered. Additionally, materials may be destroyed prior to entry into a cell. In the body, extracellular substances are subject to inactivation and/or degradation by many different mechanisms if they are not protected in some way from such a fate.
A variety of approaches have been taken to introduce biologically active materials into cells, but most of these approaches have restrictions, such as limits as to types of materials which can be transferred, which limit their usefulness. For example, nucleic acid, such as DNA, can be introduced into cells by numerous transfection techniques, many of which perturb the cell membrane chemically (e.g., calcium phosphate precipitation, DEAE-dextran, lipofection) or electrically (e.g., electroporation). Some of the chemical-mediated transfection techniques (such as lipofection, in which DNA is complexed with cationic lipids) likely involve endocytic uptake of the DNA. While these techniques are useful for introducing nucleic acid into a cell, they are not applicable to many other types of materials. Another limitation of many known approaches for transmitting materials into cells is that they are not applicable to in vivo situations, thereby requiting that the target cell be available in vitro. For example, many of the aforementioned DNA transfection techniques are useful in vitro but are not transferable to in vivo situations. Another type of technique, microinjection, can be used to introduce different types of materials into the cell cytoplasm or nucleus but is technically tedious and is limited in the number of cells which can be modified. Another approach involves using viruses, such as retroviruses, to introduce materials into cells. Viral-mediated transfer can be performed in vivo but primarily is useful for introducing DNA into cells and may not be useful for other types of materials, and, even for DNA, has limited capacity (e.g., size restrictions on the length of DNA that can be transferred). Techniques which permit transmission of materials to cells in vivo are limited and may not allow for targeting of the material to a specific cell type, which is usually desired, thus leading to the need for large systemic dosages of the material.
A delivery system which allows transmission of a variety of a biologically active materials to cells either in vivo or in vitro, protects the materials from inactivation, both extracellularly prior to delivery and intracellularly after delivery and which allows for targeting of the material to specific cells would be highly desirable for many therapeutic applications. One approach in developing such a system is to design a carrier vehicle which can carry and protect, and target if necessary, the biologically active material and which can mediate entry of the material into the cell. A possible carrier vehicle for delivering materials to a cell is a lipid vesicle. Lipid vesicles are substantially spherical structures made of materials having a high lipid content in which the lipids are organized in the form of lipid bilayers. Unilamellar vesicles have a single lipid bilayer surrounding an amorphous central cavity which can encapsulate an aqueous volume. Unilamellar vesicles can be prepared as either large unilamellar vesicles (LUVs; diameter greater than about 1 .mu.) or small unilamellar vesicles (SUVs; diameter less than about 0.2 .mu.). Multilamellar vesicles (MLVs) have many onion-like shells of lipid bilayers. Because of their high lipid content, MLVs have use for carrying certain small lipophilic molecules but have a low carrying capacity for aqueous material. Paucilamellar vesicles (PLVs) have about two-ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a central cavity free of lipid bilayers. PLVs can encapsulate both aqueous and hydrophobic material and thus can carry a wide variety of materials.
Unilamellar vesicles composed of a single bilayer of phospholipids and/or glycolipids are the most commonly used lipid vesicles for modeling of cell membrane structures since phospholipids are the primary structural component of natural membranes, including the outer cell membrane. Phospholipid vesicles have been used as carrier vehicles for delivering biologically active materials to cells. However, such vesicles do not fuse with the outer membrane of the cell but rather are generally taken up by cells via endocytosis and enter the lysosomal degradation pathway. Biologically active materials carried by the lipid vesicle may then be destroyed by lysosomal enzymes. Attempts have been made to construct phospholipid vesicles which will avoid this fate. Methods used to circumvent the lysosomal pathway include use of pH-sensitive liposomes, which fuse with endosomal membranes in the acidic environment of the endosome, thereby releasing their contents before exposure to lysosomal enzymes, and incorporation of viral fusion proteins into the phospholipid vesicle to promote fusion of the vesicle with the outer cell membrane, thereby avoiding endocytosis of the vesicle. For reviews of phospholipid vesicle-mediated transfer of materials see Mannino, R. J. and Gould-Fogerite, S., BioTechniques, 6:682 (1988); Litzinger, D. C. and Huang, L., Biochim. et Biophys. Acta, 1113:201 (1992).
The use of phospholipid vesicles as carrier vehicles for delivery of biologically active materials to cells is limited by the necessity to manipulate the vesicles so as to avoid lysosomal destruction of the encapsulated material. Furthermore, phospholipid vesicles can be costly to produce, are not stable in vitro and may not be stable long-term in vivo because of the activity of phospholipases in vivo. An alternative carrier vehicle for delivery of biologically active materials is a paucilamellar non-phospholipid vesicle. Advantages of paucilamellar non-phospholipid vesicles include that they are less costly to produce than phospholipid vesicles, are more stable in vivo than phospholipid vesicles, and have a large carrying capacity for encapsulated material. It has now been discovered that when certain paucilamellar non-phospholipid vesicles are contacted with cells they do not cause lysis of the cells and are not taken up by the cell via endocytosis, but rather fuse with the outer membrane of the cell. Thus, rather than being introduced into the endocytic pathway, and ultimately the lysosomal pathway, material carried by the paucilamellar non-phospholipid vesicle is introduced directly into the cytoplasm of the cell. Additionally, upon fusion the non-phospholipid bilayers of the vesicle are incorporated into the outer membrane of the cell.
Accordingly, an object of the invention is to provide a method of transmitting a biologically active material to a cell using a non-phospholipid vesicle carrier.
Another object of the invention is to provide a method of transmitting a biologically active material to a cell in vivo in a mammal using a non-phospholipid vesicle carrier.
A further object of the invention is to provide a method of delivering a biologically active material directly to the cytoplasm of a cell by fusion of a non-phospholipid vesicle carrying the material with the outer membrane of the cell.
A still further object of the invention is to provide a method of delivering material associated with the bilayers of a non-phospholipid vesicle to the phospholipid outer membrane of a cell by fusion of the non-phospholipid vesicle with the outer membrane of the cell.
These and other objects and features of the invention will be apparent from the following description and claims.